全柱成像毛细管等电聚焦电泳分析蛋白质药物电荷异质性

发布时间：2018-04-16 14:31

本文选题：全柱成像毛细管等电聚焦电泳 + 电荷异质性　；参考：《分析测试学报》2017年03期

【摘要】：评价了cIEF-WCID检测多肽与蛋白质药物等电点的应用效果。测定标准多肽的等电点,验证了cIEF-WCID具有高的准确度和良好的重复性(相对标准偏差0.50%)。人血红蛋白的4种主要异构体实现了基线分离;甘赖胰岛素的重复测试均只检出单一特征峰(p I 5.95±0.01);比较重组人生长激素原液和成品,等电点特征峰比例差异明显,且成品有新特征峰出现;对比进口及国产贝伐单抗,发现厂家1、3与原研药基本一致,厂家1的主成分迁移规律与原研药高度一致,厂家2的重链C末端K缺失、N末端焦谷氨酸环化或脱酰胺修饰影响了电荷异质性;通过考察尿素浓度、电解质范围和聚焦时间,优化了检测重组人促卵泡素等电点条件:2 mol/L尿素,两性电解质pH 2.5~5.0与pH 3.0~10.0按1∶1混合,聚焦电压1 000 V(1 min)~1 800 V(4 min)~2 200 V(1 min)。cIEF-WCID可快速、准确测定具有电荷异质性的蛋白类药物等电点,分辨率和重复性好,尤其是可以跟踪聚焦过程中样本的迁移特征,特别适合蛋白类药物复杂电荷异质性的检测。
[Abstract]:The application effect of cIEF-WCID in detecting the isoelectric point of polypeptide and protein drug was evaluated.The determination of the isoelectric point of the standard polypeptide proved that cIEF-WCID had high accuracy and good repeatability (relative standard deviation was 0.50%).The four main isomers of human hemoglobin were separated from baseline, and only a single characteristic peak (P I 5.95 卤0.01) was detected in repeated test of insulin of kale, and the ratio of isoelectric characteristic peak was significantly different between the recombinant human growth hormone solution and the finished product.Comparing with the imported and domestic bevacizumab, it was found that the principal component migration law of manufacturer 1 was basically consistent with that of the original drug, and the principal component migration law of manufacturer 1 was consistent with that of the original drug.The cyclization or deamination of the N-terminal pyroglutamic acid in the heavy chain C-terminal K deletion of manufacturer 2 affected the charge heterogeneity, and the concentration of urea, the electrolyte range and the focusing time were investigated.The isoelectric point conditions for detection of recombinant human follicle stimulating hormone (FSH) were optimized. When the amphoteric electrolyte pH 2.55.0 was mixed with pH 3.0 10. 0 at 1:1, the focusing voltage of 1 000 VX 1 min)~1 800 VX 4 min)~2 200 VX 1 min).cIEF-WCID was rapid.Accurate determination of the isoelectric points of protein drugs with charge heterogeneity has good resolution and repeatability, especially it can track the migration characteristics of samples during the focusing process, and is especially suitable for the detection of complex charge heterogeneity of protein drugs.
【作者单位】：北京正旦国际科技有限责任公司;北京蛋白质组研究中心蛋白质组学国家重点实验室国家蛋白质科学中心(北京);江汉大学护理与医学技术学院;北京绿绵科技有限公司;军事医学科学院放射与辐射医学研究所;
【基金】：国家高技术研究发展计划863项目(2014AA020906) 国家科技部重大仪器专项(2012YQ18011710) 蛋白质组学国家重点实验室课题(SKLP-O201412)
【分类号】：TQ460.72;O657.8

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